Recent Advancements in Biomaterials for Chimeric Antigen Receptor T Cell Immunotherapy

Cellular immunotherapy is an innovative cancer treatment method that utilizes the patient’s own immune system to combat tumor cells effectively. Currently, the mainstream therapeutic approaches include chimeric antigen receptor T cell (CAR-T) therapy, T cell receptor gene-modified T cell therapy and chimeric antigen receptor natural killer-cell therapy with CAR-T therapy mostly advanced. Nonetheless, the conventional manufacturing process of this therapy has shortcomings in each step that call for improvement. Marked efforts have been invested for its enhancement while notable progresses achieved in the realm of biomaterials application. With CAR-T therapy as a prime example, the aim of this review is to comprehensively discuss the various biomaterials used in cell immunotherapy, their roles in regulating immune cells, and their potential for breakthroughs in cancer treatment from gene transduction to efficacy enhancement. This article additionally addressed widely adopted animal models for efficacy evaluating.


Introduction
Cellular immunotherapy is an emerging cancer treatment ap proach, which harnesses the patient's own immune system to target and destroy malignant cells [1,2].Since the U.S. Food and Drug Administration approved chimeric antigen receptor T cell (CAR-T) cell therapy for B cell lymphoma in 2017, the development of cellular immunotherapy, encompassing CAR-T therapy, T cell receptor gene-modified T cell (TCR-T) therapy, and chimeric antigen receptor natural killer-cell (CAR-NK) therapy, has made significant breakthroughs and demonstrated a great potential in clinical practice [3].
CAR-T therapy involves modifying the patient's own T cells to express specific receptors capable of recognizing and attacking malignant tumor cells [4,5].This innovative approach has demonstrated outstanding efficacy, particularly in addressing hematological malignancies like acute lymphoblastic leukemia and non-Hodgkin lymphoma [6,7].TCR-T therapy entails genetically altering the patient's T cells to express T cell receptors (TCRs) that can recognize particular tumor antigens, thereby identifying and combating tumor cells [8,9].While still in the research and clinical trial stages currently, TCR-T cell therapy has shown promise in treating some solid cancers, including melanoma and colorectal cancer [10].
CAR-NK therapy is an emerging field in cellular immunotherapy that utilizes engineered natural killer cells (NK cells) to treat cancer [11][12][13].CAR-NK therapy has a number of potential benefits over CAR-T therapy.Firstly, CAR-NK cells exhibit relatively more temperate anticancer effects [14,15], for the reason that CAR-NK cells have less propensity to trigger intense, overwhelming, and excessive immunological responses, which contributes to a decrease in treatment-related adverse effects [16].Finally, NK cells have a puissant potential of proliferation, which makes them easier to produce and manufacture in large quantities in vitro [17].While holding promising prospects, both therapies are currently in a nascent stage of development with relatively modest scale in biomaterials researches.Therefore, this review will illustrate the biomaterials application in cellular immunotherapy with CAR-T therapy as a representative case.
With major advancements in the treatment of cancer, cellular immunotherapy is currently considered as an important therapeutic strategy [2].However, this treatment approach still faces some challenges, such as the durability of treatment responses, side effects associated to the treatment, the longevity of therapeutic outcomes, and costs [18,19].Furthermore, its effectiveness against solid tumors has been more challenging due to the complex tumor microenvironment (TME) and issues related to penetration and persistence of the engineered cells within solid tissues [20].Moreover, there is a risk of on-target, off-tumor effects, where healthy cells expressing the targeted antigen are inadvertently attacked, which may lead to side effects and complications [21].
Conventional manufacture procedure of CAR-T can be elucidated as the following process: the deviation of lymphocytes through leukapheresis, target gene transduction, activation and expansion of CAR-Ts, quality control, and final administration.Dependent of in vitro storage and reaction conditions, the aforementioned steps are rather time-consuming and highcost.Unfortunately, each procedure is confronted with unforeseeable risks and challenges.For example, a great proportion of patients with malignancy were used to receive chemotherapy, which added difficulties to collecting sufficient leukocytes for subsequent treatments.Other disadvantages incompletely include functional aberration during storage, exhaustion under in vitro activation, cytotoxicity brought by traditional transduction, and cell intolerance to TME [22][23][24].Facing these shortcomings, appropriate development of biomaterials offer assistance in every procedure such as bio-instructive implantable scaffolds that allowed in vivo transduction and release of CAR-T to extensively shorten the manufacture time [25] and nanomaterials for normalizing the TME [26].The more comprehensive application of biomaterials will be illustrated below.
Although the development of novel cellular immunotherapy strategies, such as TCR-T therapy and CAR-NK therapy, partially solves these problems, efficacy remains unsatisfactory.Biomaterials have become essential components in cellular immunotherapy, fulfilling various roles such as carriers, scaffolds, or supplementary therapeutic tools to enhance treatment effectiveness and support the functionality and longevity of engineered immune cells [27].As effective carriers for CAR-T therapy, biomaterials are not only offering a favorable 3-dimensional (3D) milieu microenvironment for engineered T cells but also reducing nonspecific immune responses, hence ameliorating treatment-associated adverse effects and improving overall safety and efficacy [28].Furthermore, by implementing strategic alterations, researchers have the ability to employ these substances for the purpose of transmitting immune regulatory factors or tumor-specific antigens [29].This process ultimately enhances the precision and effectiveness of cellular immunotherapy [30].
In recent years, the study of employing biomaterials to enhance the efficacy of cell immunotherapy has advanced significantly [27,[31][32][33][34].With the purpose of objectively presenting current progresses, highlighting both innovations and shortcomings of relevant researches and serving possible future development directions of biomaterials, we cover a wide spectrum of biomaterials utilized in cell immunotherapy with primary emphasis on CAR-T and elucidate their functions in regulating immune cell proliferation and activation as well as facilitating more accurate and efficient cell immunotherapy in this comprehensive review.In the process, we focused on the role of different types of biomaterials in gene transduction, efficacy monitoring, and therapy efficacy enhancement.We also present a thorough overview of several cell carrier platforms and animal models used for evaluating the therapeutic effects in order to assist further investigations in the area of biomaterialenhanced cell immunotherapy.Ultimately, we will provide our perspectives on the future of biomaterials in immunotherapy.In conclusion, the future of biomaterials in the realm of cell immunotherapy is exceedingly promising, and we anticipate significant breakthroughs in the treatment of cancer through the integration of biomaterials with cell immunotherapy.Moreover, with the progress of new cellular immunotherapy research, such as TCR-T cell therapy and CAR-NK cell therapy, we are also looking forward to the further integration and application of biomaterials for a wider range of cellular immunotherapy.

Gene Transduction
Gene transduction is considered as a crucial step in the manufacture of CAR-T [35].The effective and precise transduction of CAR gene allows engineered T cells to specifically target tumor cells while minimizing potential harm to healthy tissue [36].A delivery system that can well condense transduction efficacy, ensure biosecurity, and maintain cost-effectiveness is urgently needed in order to ease the widespread clinical implementation [37].As Table 1 showed, a range of strategies have been unearthed based on distinct principles and materials [38,39].Currently, prevailing tactics utilized can be broadly categorized into 2 components: ex vivo and in vivo.In this review, we provide a concise overview of both methodologies, with a particular focus on the application of biomaterials within this field.
Electroporation serves as a fundamental technique for transducing CAR gene by physical means, playing a pivotal role in diverse delivery platforms [48].In the configuration established by bulk electroporation, the conducting buffer solution contains the suspended cell and gene carrier between 2 connecting electrodes.The cell membrane, being an electrical insulator with stable transmembrane potential, is capable of being influenced by an external electric field [49].This external electric field can cause an increase in the potential difference across the membrane, reaching a threshold that leads to breakdown of membrane [48].Consequently, temporary pores on membrane are formed enabling the diffusion of gene vectors [50].Electroporation is a highly efficient method with clinical transformation value, capable of achieving high throughput, regardless of the constraints imposed by cell type, cell cycle, or biological characteristics of carriers [51].The transposon system in mRNA have been extensively investigated as approaches for gene transduction in conjunction with electroporation [52][53][54].As for transposon systems, the Sleeping Beauty and PiggyBac are relatively stably established [53].Figure 1A showed schematics of the hybrid adeno-associated vector (AAV)-SB construct, SB100X mRNA electroporation, and CAR T/NK/macrophage/ induced pluripotent stem cell (iPSC) generation [55].Combined with gene-transduction techniques, human iPSC is capable of converting into patient-specific progenitor or functional cells even tissue and organs, among which CAR-T involved.The whole procedure for iPSC-related CAR-T study is composed of iPSC preparation, CAR transfection, and differentiation [56].Ini tially, somatic cells principally extracted from peripheral blood are subject to reprogramming triggered by virus-mediated delivery or nonintegrating gene transfection and then reverse into iPSCs [57,58].Subsequently, CAR gene is introduced into iPSCs through various pathways including lentivirus [59,60], electroporation, with zinc-finger nuclease editing [61] or PiggyBac transposon systems [62].CAR-bearing iPSCs are then cultivated within a specially designed culture medium and exposed to selected growth factor and cytokines that guide their predetermined differentiation pathways following strict timing.To date, researchers have provided detailed protocols for iPSC-NK [63], iPSC-T [59], iPSC-macrophages [60], and iPSC-monocytes [64].These systems exhibit enhanced cargo capacity and biosecurity as they are proficient in mediating steady integration of DNA into the genome of the host.In the meanwhile, it is worth noting that these transposon systems maintained as plasmid DNA demonstrates cost-effectiveness in terms of manufacturing and holds promise for potential scale-up production [65].The RNA electroporation enables the production of the CAR construct for a period of 7-d cycle, thereby establishing a basis for further ex tensive examination of single-chain variable fragments and the safety of CAR constructs [66,67].Nevertheless, electroporation has faced significant criticism due to its possible cytotoxicity, including membrane damage, lipid peroxidation, and high-rate cell death [68].These effects are believed to be caused by inappropriate physical parameters used during the process.
Fig. 1B demonstrated a schematic overview of intracellular delivery by membrane permeabilization with photothermal nanofibers [69].Photoporation could serve as an alternative, operating on the similar concept of membrane disruption [70].Photothermal sensitizers, such as gold nanoparticles (AuNPs), have the ability to transform light energy into thermal energy, causing the reduction of water evaporation in the vicinity and the creation of vapor nanobubbles [71].The vapor nanobubbles rapidly undergo collapse, resulting in the generation of shockwaves that induce the formation of reversible pores in the membrane [70,72,73].To address the issue of nonbiodegradability of AuNPs, a team led by Braeckmans developed biocompatible polymeric nanoparticles as a potential solution.They synthesized the polydopamine nanoparticles as a substitute for AuNPs and coated the surface of polydopamine nanoparticles with bovine serum albumin to booster the colloidal stability and cell affinity.Following laser irradiation, the effective transduction of mRNA expressing FD500 and enhanced green fluorescent protein was observed in human T cells, HeLa cells, and Jurkat cells [74].It is worth mentioning that the collaborative integration of electroporation and microfluidic systems can contribute to the generation of higher localized and concentrated electrical fields.It becomes feasible by the utilization of controllable microfluidic channels, which in turn promote cell viability [75].
Other methods may potentially be employed for CAR-T gene transduction.Magnetofection is a promising approach for application in CAR-T therapy, with a close association to biomaterials.It enables the complex composed of nucleic acids and magnetic nanoparticles to be absorbed by endocytosis or pinocytosis under the guidance of external magnetic field [76].Recently, bacterial magnetic particles (BMPs) have been displayed for future application.Fang et al. conducted research showing that the BMPs-PEI gene carrier exhibited higher transfection efficiency in Cos-7, HeLa, and HepG2 cell lines.This vector system is biocompatible with low cytotoxicity when delivering DNA into primary cells [77].

In vivo components
The conventional ex vivo strategy involves the extraction of T cells from the blood of patients, followed by a series of steps including activation, transduction, expansion, and ultimately autologous reinfusion.The production of these products is characterized by a high degree of customization, necessitating labor-intensive, time-consuming, and expensive industrial engineering processes.The technology is also facing criticism due to certain flaws.One such flaw is the potential transfer of CAR into cancer cells, which can occur when these cells infiltrate the bloodstream and evade detection by T cells.
The utilization of in vivo methods for gene transduction has developed for therapeutic purposes.Complex procedures have been circumvented in favor of an exclusive reliance on particle infusion.To a significant extent, the occurrence of side effects, such as graft-host reaction and reduced viability of CAR-Ts, was effectively mitigated.Vectors that are commonly employed encompass both viral and nonviral types.The distinctions be tween nonvirus and virus are enumerated in Fig. 1A and B.
Both of these approaches have a shared basic principle, which is to provide the carriers with the ability to selectivity target certain subsets of immune cells.The identification of specific cell markers is a commonly employed approach, with the use of specialized materials or structures such as single-chain variable fragments or designed ankyrin repeat proteins enhance stronger affinity to particular subsets.The viral-vector field is greatly concerned with retrovirus families, such as lentiviral vectors (LVs), γ-retrovirus, and AAVs.Retroviruses has the capacity to do reverse transcription, wherein the viral RNA genome is converted into complementary DNA (cDNA).This cDNA is then integrated into the chromosomal DNA of the host organism [67].
LVs are widely employed in clinical research, constituting approximately 54% of the methods to induce T cell activation.The determination of transduction preference, also known as tropism, is governed by the viral envelope protein, which is intricately associated with the process of pseudo typing.As for LVs, the replacement of the surface protein with glycoprotein G derived from vesicular stomatitis virus enables the internalization of LVs by various categories of cells through the mediation of low-density lipoprotein receptor.Conversely, γ-retroviral vectors have the ability to mimic the amphitropic murine leukemia virus as well as comparable approaches [67].Both γ-retrovirus and lentivirus have been found to contribute to increased expression and improved transduction efficacy [78].
Additional groups of viruses were also documented.AAVs, which are nonenveloped viruses containing single-stranded DNA, enter cells through a receptor-mediated mechanism.Through endosomal escape, synthesis of second-strand DNA (ds DNA) also enters the nucleus by a process of endosomal escape.Once inside, it undergoes the synthesis of ds DNA.Subse quently, the ds DNA has the potential to either integrate into the genome or persist as an epistome [79].Compared with LVs, the transduction span of the vector in activated lymphocytes was notably shorter.Capsid engineering is a frequently employed method of modification.In a recent study, researchers successfully achieved efficient transduction in preclinical models of Tractargeted CAR-Ts by employing a developing AAV variation [80].
Although virus-based vectors exhibit remarkable transduction efficiency, it is still hindered by certain shortcomings.The immunological response of the host was still likely to be triggered by all viral proteins.Due to gene integration behavior, clinical monitoring process should place a premium on risks related to intersectional oncogenesis.Different types of viruses exhibit distinct preferences for insertion, as seen in patients who were exposed to lentivirus-mediated transduction.In these cases, the lentivirus tended to insert itself into the introns of genes that were actively involved in transcription [67].Consequently, these patients experienced reduced T cells expansion and insufficient antitumor efficacy [81].Simultaneously, it should be noted that the aforementioned viral vectors exhibited a constraint in terms of payload capacity, in particular limited to 8 kb which poses a challenge for supplementary genetic manipulations.

Nonvirus delivery platforms
Nonvirus delivery platforms rely extensively on biomaterials, including lipid, polymers, and inorganic nanoparticles, which are customized based on the specific properties of the materials and practical requirements.Herein, the nonvirus delivery platforms are classified into 2 categories, including organic and inorganic, in order to provide a more comprehensive understanding.

Polymers
The development of polymer-based carriers involves the utilization of both electrostatic and covalent interactions [82].Extensive research has been conducted on polycations.These synthesize of these polycations is principally achieved using polyethylenimine (bPEI) [83], poly(2-dimethyl) aminoethyl methacrylate) (pDMAEMA) [84], polyamidoamine (PAMAM) [85,86], and poly (β-amino ester) (PBAE) [87].The polycation can be synthesized into different geometric configurations, such as linear-branched (comb), cyclic-branched (sunflower), or starshaped.According to the findings, variations in transduction effectiveness and cell viability can be attributed to differences in core size, branch length, and numbers.Nonetheless, it is important to note that a majority of polycations still fail to achieve a satisfactory transfection efficiency [88].Furthermore, there exists an extra mentality of designing based on polymer.
Stephan et al. made significant contribution to this field.They developed an innovative nanoparticle in order to achieve the in situ reprogramming of T cells.The anti-CD3e f(ab')2 fragments were conjugated to the surface of PBAE nanoparticles to specifically target to the T cells.The plasmid DNA encoding 194-1BBz CAR, which were aimed at leukemia cancer cells, and iPB7 transposase derived from PiggyBac transposase system were encapsulated altogether into nanoparticles.To reduce the occurrence of nontarget combinations, a negatively charged polyglutamic acid was introduced as a coating agent for nucleic acids.The utilization of bioluminescence imaging facilitated the observation that the CAR gene was effectively transferred to CD3 T cells precisely, as evidenced by the detection of coexpress click beetle red luciferase.In addition, the longevity of mice and the growth of tumors demonstrated a comparable antitumor capability to that of traditional ex vivo engineered CAR-Ts with minimal toxicity [89].In 2020, this team successfully achieved nanoparticles as a means to deliver mRNA.Similar to previous structure, the nanoparticle is comprised of PBAE that encapsulates mRNA, while concurrently including a target ligand CD8 in conjunction with poly (glycolic acid) (PGA), as shown in Fig.
2A [90].A series of findings demonstrated successful delivery of mRNA by the nanoparticle, resulting in sustained CAR expression for 1 week following each injection.The survival duration of mice with prostate tumors was prolonged for 40 d, and effective infiltration of CAR-T into solid tumor was found by fluorescence imaging.It is hypothesized that the persistence of tumors can be attributed to their down-regulation of target antigens.The nanoparticle simultaneously displayed the ability of intracellular HBV core antigen recognition while delivering TCR transgene.In contract, most in vitro transcribed mRNAencoding CAR were only capable of recognizing antigens produced on the cell surface.

Lipid
There are further studies that investigate the potential utilization of lipid nanoparticles.The production of polymer nanoparticles often exploits the utilization of electrostatic connections between lipid molecules with positive charges and nucleic acids with negative charges.This interaction leads to form a complex, which is later internalized by cells and releases its cargo.It has been found that reengineered T cell immunotherapy holds promise for the reversal of cardiac fibrosis [91].Figure 2B demonstrated an orthogonal design of experiments for optimization of lipid nanoparticles for mRNA engineering of CAR-Ts.Lipid nanoparticles (LNPs) consist of 4 main components: ionizable lipid, cholesterol, phospholipid, and PEGylated lipids [92].Genetic materials including small interfering RNA (siRNA), mRNA, and plasmid DNA [93] are encapsulated into the mixture of components mentioned above through methods like microfluid mixing [94].Take the LNP siRNA system as an example; after venous administration, it will combine with ApoE and then enter the hepatocytes through endocytosis mediated by ApoE-related ligands17 and 19.Under the pH environment of endosomal, large proportion of ionizable lipid will be protonated and attracts the endogenous anionic lipid which leads to the formation of endosome-disrupting nonbilayer structures and release of contents.It is worthwhile to mention that the LNP can be targeted toward various specific cells or tissues as long as equipped with therapeutic proteins including monoclonal antibodies.Epstein.et al. created an experimental immune therapy utilizing in vivo transduction for the treatment of cardiac fibrosis [94].The LNP was coupled with a protein that selectively binds to CD5, a molecule that plays a vital role in cellular processes.This protein is loaded with mRNA encoding CAR gene, which specifically targets fibroblast activation protein and Cre recombinase.Bioluminescence imaging indicated that only the experimental group, which received the injection of CD5/LNP-FAPCAR, exhibited detectable and consistent CAR-T subsets.The echocardiography surprisingly showed a notable improvement in cardiac function, namely in the left ventricle diastolic function, following the administration of temporary CAR-Ts [95].However, due to the disability of genomic integration, mRNA was restricted to cytoplasm and prone to be diluted or be decomposed, triggered only transient expression of fibroblast activation protein.
By considering the existing methods employed for in vivo gene delivery, it is foreseeable that the efficient generation of targeted CAR-Ts will eventually be achieved.There remain some unresolved issues pertaining to in vivo transduction that warrant significant attention for future improvement.The efficacy of in vivo transduction is currently impeded by off-target effects, which occur when subsets of cells expressing the same markers are mistakenly targeted, resulting in an insufficient number of effective CAR gene deliveries and an increased risk of unexpected immune functional interference.In contrast to the conventional ex vivo transduction approach, the utilization of in vivo transduction results in a significantly lower production CAR-Ts by many orders of CAR-Ts by many orders of magnitude.This reduction can be attributed to factors such as gene dilution, degradation, or inactivation.Immune reaction to the carrier or its binding protein, as well as the process of phagocytosis, has a significance in reducing carrier efficacy.In clinical practice, it is recommended to administer subsequent injections, as this helps to sustain an optimal concentration level and reduce the likelihood of tumor reoccurrence.Despite that, it is likely that insertional mutagenesis occurs in the most significant integration systems.We have yet to make significant progress.

Other materials
Actually, in addition to common vector based on lipid or cationic polymers, other attempts have been made to explore more potential transfection tools.The cell penetrating peptides (CPPs) is the first to mention.Researchers found that CPP with am phipathic RALA motif repeat could deliver mRNA into dendritic cells (DCs) [96].However, the mechanism underlying remains unknown and is considered to relate to micropinocytosis or lipid bilayer disruption [97].Besides, nanostructure, which encompasses nanostraws, nanowires, and so on, is representative of one alternative to directly pierce the cell membrane and transfer nuclei acid.It embraces various cargos including siRNA, pDNA, and other molecules [98].Shokouhi et al. developed a nonviral electroactive nanoinjection platform.Configured under low voltage, the electroactive nanotubes enabled surprising delivery efficacy and expression with cell viability maintaining high-level [99].Such combination gives a possible answer to overcome the disadvantages of electroporation and nanostructure.It is of note that both methods mentioned above still lack a clear mechanism and suffer from the disadvantage of low transfection efficacy.Other research reported recombinant PP7 virus-like particles, a vector offering more stable properties and protecting the mRNA from degradation compared to CPPs [100].and efficacy of transfected cells [102,103].Combined with biomaterials, researchers offered a different vision of better transfection.Liu et al. synthesized a novel superparamagnetic nano-sized iron-oxide particle, IOPC-NH2 series, to increase safety.The particles are encapsulated in polyethylene glycol (PEG), which lacks of immunogenicity and antigenicity.Without the use of the aforementioned transfection agents, the particle demonstrated a transfection efficiency of over 90%.No discernible difference was found in the expression of CD62L and CD25 between IOPC-NH2 labeled and unlabeled normal rat T cells in the rat model.Additionally, the immune response toward LPS stimulation was identical in IOPC-NH2 labeled Jurkat cells and unlabeled Jurkat cells, indicating the hypotonicity of the particle [104].

Monitoring the Efficacy of CAR-T
Based on the prior research on the incorporation of endothelial progenitor cells labeled with ultrasmall superparamagnetic particles of iron oxide (USPIOs), Zhang et al. developed the amino alcohol derivatives of glucose-coated nanoparticles.These nanoparticles were utilized to track the infiltration and duration of CAR-Ts in a manner with noninvasive procedures.The optimized concentration of iron, specifically 37.5 μg/ml, was selected based on the comparatively high viability of CAR-Ts.The transmission electron microscopy analysis indicated the presence of black particles in the cytoplasm of CAR-Ts that were labeled with USPIO, in contrast to the nonlabeled groups.This observation suggests that the complexes exhibit a high level of effectiveness in terms of endocytosis [103].Furthermore, the application of subsequent CD11b and Iba-1 immunostaining effectively excluded the possibility of nonspecific endocytosis.MRI was employed to monitor the growth of glioblastoma multiforme (GBM) xenografts, Susceptibility-weighted imaging (SWI) was performed on days 3, 7, and 14, revealing the progressive emergence of hypointense signal following the injection of a minimum of 1 × 10 5 labeled CAR-Ts.Non labeled cells showed no hypointense signal.The diffusion-weighted imaging MRI imaging revealed a noteworthy drop in the Ktrans value and a substantial increase in apparent diffusion coefficient in USPIO-labeled CAR-Ts, as compared to the control group, when administered after 3 d.This finding suggested that the early stages of vascular and cellular changes can be detected using these imaging modalities [104].This discovery was further substantiated by subsequent histological sections and SWI imaging.Additional contrast agents (e.g., fluorine-19 perfluorocarbon) have been reported with a promising potential for future applications.This is due to their low toxicity and negligible interference with background signals.The comparison between MRI and positron emission tomography (PET) is presented in Table 2.
Nuclear medicine imaging has also been extensively explored, with the direct and indirect labeling systems emerging as the 2 primary methods.Prior to the emergence of PET imaging, significant efforts were focused on the application of single-photon emission computed tomography for T cell direct-labeling tracking.The radionuclides 111In and 99mTc were frequently used for ex vivo cell tracking but without enough labeling stability.111In-oxine and 99mTc-HMPAO solved the problem relatively by promoting the diffusion into cells and combination of isotopes and protein in cytoplasm.However, the requirement for a significant increase in dosage due to the sensitivity and loss through efflux of radioisotopes demanded huge dose of addition, which reduced to the cytotoxicity.The presence of photon attenuation and scattering posed a significant challenge in accurately quantifying the signal intensity.The all above the defects hindered the pace of further advancements in single-photon emission computed tomography.In contrast, PET has emerged as a viable alternative due to its superior tumor background ratio.The underlying principle of PET involves the emission of radionuclides, which subsequently produce positrons and neutrinos in order to maintain stability.Following the annihilation of a positron with an electron, the resulting photons are emitted and subsequently detected by specialists.To address the issue of the relatively short half-life of radionuclides, researchers have explored the use of chelating agents, such as methyl 4-methylbenzene-1-sulfonate (PTSM), to slow down the rate of decay.However, this approach still faces challenges because of the significant efflux of isotope.
Harmsen et al. designed a novel nongenomic imaging technique utilizing dual-modal PET/near-infrared fluorescent (NIRF) nanoparticles to obtain a sustaining observation in vivo for 1 week following immune cell infusion, as shown in Fig. 3A.By incubation with the NIRF silica nanoparticle labeled with 89Zr as well as protamine and heparin, the CAR-Ts were administrated peritoneally and intravenously.PET showed the concentrated signal in the lungs and liver following intravenous delivery, and in the liver and spleen following peritoneal administration.The imaging results obtained on day 14 using NIRF demonstrated a strong correlation with PET.Furthermore, the technique employed to mark the CAR-Ts demonstrated significant clinical translation potential, as a majority of these methods have been validated for clinical application and are already in widespread usage.Never theless, the nanoparticle continued to experience apoptosis following a 1-week infusion.However, it has been observed by researchers that the preceding analysis of the peritoneal tumor section has demonstrated that the nanoparticle released was effectively internalized by tumor cells, indicating that this system has the potential to be applied to better nanodrug delivery [105].The process of indirect labeling necessitates modifications to genetic materials, which facilitates the production of certain reporter proteins.The reporter gene provides enhanced stability in expression and extended duration of monitoring.The hindrance of transduction-related risks on cell functionality and immunogenicity, along with the ethical and moral concerns, has unfortunately impeded its advancement.
In vivo optical imaging encompasses several techniques, including as fluorescence imaging, bioluminescence, confocal and 2-photon microscopy imaging, as well as diffuse optical tomography.The categorization system can also be broadly classified into direct and indirect.The direct labeling system requires the combination between dye and cell, such as the near-infrared (NIR) dye DiD system used for labeling NK-92 cells and the avidin-biotin system.The indirect system necessitates the alteration of the genome.However, the research on the application of biomaterials in optical imaging is limited.Furthermore, additional attempts of monitoring CAR-T have been undertaken.Popovtzer et al. developed an innovative and versatile nanoprobe by using PAMAM dendrimers as carriers, integrating AuNPs with a fluorescent calcium sensor, and forming a dual-modal nanoprobe.The hyperbranched nano polymers, known as 5 PAMAM, exhibited a modifiable structure based on the applied load.AuNPs have been widely regarded as a desirable contrast agent for computed tomography (CT) due to excellent x-ray attenuation, and many studies revealed the correlation between the activation of T cells and intracellular calcium ion concentration increase.The examination con ducted by researchers using enzyme-linked immunosorbent assay revealed that the labeled CAR-Ts exhibited comparable tumor necrosis factor-α secretion ability and immunogenicity in comparison with control groups.The results of in vivo fluorescent imaging indicated that the fluorescent signal peaked at 4 h after administration and remained detectable for nearly 24 h.This temporal profile of fluorescence provides valuable insights into the activation of T cells.As shown in Fig. 3B, the CT scan conducted 24 h after administration demonstrated that T cells labeled with nanoprobe showed a remarkable stronger signal compared to nonlabeled cells.This signal strength effectively displayed the distribution of engineered cells [106].
In overall, the monitoring methodologies of tracking CAR-Ts demonstrate substantial potential.However, there remain certain issues that require attention, for example, the signal attenuation caused by cell lysis or exocytosis, which hinder the feasibility of prolonged observation.There is still a significant distance to be covered in order to achieve a stage where a meth od can be considered safe, accurate, real-time, and cost-effective.

Efficacy Enhancement
In recent years, biomaterials have been increasingly used as effective carriers in cellular immunotherapy for CAR-T therapy and yielded good results, as shown in Table 3.

Nanoparticles and microparticles systems
Nanoparticles and microparticles, which are extensively studied biomaterials, play a crucial role in enhancing the effectiveness of tumor eradication.The particles possess the capacity for adjustable size and deformability, which may be customized to meet various tissue-infiltration needs and unique physical and chemical properties.This characteristic serves as a basis for materials development, as it is derived from a wide range of sources.The charge effect is a significant factor that impacts the absorption by antigen presentation cells, subsequently resulting in the activation of CAR-Ts.In addition, surface functionalization is an effective technique that may be utilized to achieve targeted cell binding.To provide a concise overview, extant studies pertaining to this subject matter can categorized into organic-based and inorganic-based.It is important to note that in practical applications, different materials often combine with one another to create a convergence of distinct advantages.The effect is of major concern because it influences antigen-presenting cell uptake, which leads to CAR-T activation downstream.Furthermore, surface functionalization, as a powerful tool, can fulfil the targeting of certain groups of cells while also serving as a crucial design thrust.To provide a concise overview, existing researches relevant are classified into organic and inorganic categories.However, it should be noted that in practice, multiple materials actually combine to create unique advantages [103].

Organic materials
The polymers-based biomaterials dominate in terms of numbers developed.Diversified materials provide more complex structures, surface functional units, and special qualities to produce desired results, such as controlled drug release and degradation in response to a specified environmental pH value.Conversion under TME circumstances, extended retention, and reduced biotoxicity could be feasible design foothold in the quest to enhance therapeutic efficacy.All of these traits contribute to transformation of the entire CAR-T therapy process to our advantage, from enhanced cell expansion and activation to a change in the harsh TME, and make polymer-based biomaterials increasingly appealing.Kim together with his team collaborated to develop a working integrated nanosystem.The nanosystem contained nanoparticles loaded with doxorubicin, as well as biodegradable photobleachingresistant fluorescent polymer (BPLP)-polylactide copolymers (BPLP-PLAs).These copolymers were equipped with pH-sensitive linkers, allowing them to attach to the surface of T cells through click chemistry.Upon exposure to the mild acid TME, the linkers underwent cleavage, resulting in the dissociation of nanoparticles loaded with doxorubicin from CAR-Ts.Subse quently, these nanoparticles initiated a controlled release of medicines, therefore augmenting the cytotoxic effect.The subsequent investigations provided justification for the significant protection of normal cells by specific attachment of nanoparticles to CAR-Ts using bio-orthogonal click reaction.The inclusion of BPLP-PLAs in the system resulted in the simultaneous acquisition of an additional modality of imaging [107].Other studies have also observed a comparable structure with a distinctive mechanism for enhancing the antitumor efficacy.Duwa et al. pioneered the development of nanoBiTEs.It involved poly (lactic-co-glycolic acid) nanoparticles loaded with R848 and afterwards coupled with anti-CD3 and anti-programmed cell death ligand 1 (PD-L1) antibodies.Extensive research has been conducted on poly (lacticco-glycolic acid) nanoparticles, which have been demonstrated to possess biodegradability, biocompatibility, and the ability to undergo easy surface modification.These bispecific nanoparticles effectively achieved local immune-stimulation using a reinforcement strategy which referred to blocking of immune check point and promoting the DCs maturation [108].
The inherent variability and modifiability of polymer structures enable the effective integration of many therapeutic components, resulting in an outcome that exceeds the cumulative effects of each individual constituent.Phototherapy encompasses 2 modalities, namely photothermal therapy and photodynamic therapy.The photothermal therapy method causes cell death by increasing the temperature of the immediate surroundings, taking advantage of the vulnerability of cells to heat.On the other hand, the photodynamic therapy technique depends on the generation of the reactive oxygen species to induce cell death [109].The effectiveness of phototherapy has been constrained due to the absence of safer and more efficient photothermal agents with higher tumor cell selectivity.Poly meric nanoparticles have been well recognized as promising photothermal agents due to their high light absorption, effective photothermal conversion, and accurate selectivity [110].The team led by Cai reported a study on indocyanine green nanoparticles engineered CAR-T biohybrids.Indocyanine green is a classical NIR absorbing sensitizer with excellent photo thermal conversion efficiency [111].Based on the findings from immunofluorescence imaging and other experimental outcomes, it was determined that integration of mild photothermal intervention with indocyanine green nanoparticles effectively led to the remodeling of the TME and facilitated the recruitment and infiltration of CAR-Ts.Notably, the enhanced clearance of tumors was observed [112].
The utilization of polymer-based nanoparticles is a desirable approach for tailoring therapeutic interventions in response to the complex and individualized conditions of the innate environment in patients.The emerging field of biomimetic nanotechnology and glycoengineering represents a significant advancement in engineering capabilities.However, it is advised that the biosafety and biodegradation of organic reagents and industrial production should be thoroughly assessed to prevent the potential accumulation of cytotoxicity [103].
With the exception of magnetic materials, lipids dominate in nanocarriers development.Considered as nontoxic and biodegradable with low immunogenicity, the materials have several advantageous properties, including the ability to undergo extensive surface modification, size regulation, and adaptive encapsulation.These differentiating traits include both hydrophobicity and hydrophilicity, which are beneficial for accommodating different types of payloads.It also possesses a low production cost and utilizes scalable production technique [113,114].The administration of immunoregulation medications to specific tumor sites is a well-known approach in reversing the TME.Among the several ways employed, lipid-nanocarriers have emerged as the preferred alternative due to their desirable properties.One exemplary instance is proposed by a research team led by Irvines.They utilized the immunoliposomes to deliver the small molecule, namely the inhibitor of transforming growth factor-β.Transforming growth factor-β is known as an immunosuppressive cytokine and is related with the activation of cancer-associated fibroblasts [115].The purpose of utilizing immunoliposomes was to modify the TME.PEG was used as a coating agent for the liposome to hinder any potential interaction between the lipid layer and plasma proteins [116].Conventional lipid materials administered intravenously are criticized for short residence period in circulation because of macrophage phagocytosis [117].PEG molecules could provide liposomes with protective hydrophilic layer thus reducing the aggregation and interaction with blood components, which increases the period in circulation.However, pegylation also bring about unsatisfactory influences, including undermining the efficacy of targeted cell recognition and endosome escape [118].Additionally, previous experiments reported that lipidbased nanoparticles was associated with the activation of complement system through alternative and classic pathways and finally resulted in pseudoallergy, infusion reaction, and acute hypersensitivity [119].There is also growing concern about the reproducible fabrication formulation of lipid nanoparticles since minor deviation could lead to drastic change of properties in terms of large-scale standardized production [116].The similar structure is seen in Stephen's study as well.They successfully synthesized nanoliposomes that were coated with tumor-targeting peptide iRGD on their surface.These nanoliposomes were designed to encapsulate a phosphoinositide 3 kinase inhibitor, which acts as an immunosuppression, and a-GalCer agonist, which functions as an immunostimulant.The results showed enhanced homing efficacy, prosperous cell proliferation, and reduction in tumor size [120].
Organic biomaterials derived from lipids can manifest in various forms, outside the scope of nanoparticles.Considering the injectable lipid gel (LG) as an illustrative example, Luo et al. developed an integrated drug delivery system, known as the mild photothermal-sensitized immunotherapy system, in which an injectable LG plays a crucial role.The LG is composed of a combination of soybean phosphatidylcholine and glycerol dioleate, which have been deliberately manipulated to induce a reversible morphological alteration.By incorporating the photothermal agent IR820 and the anti-PD-L1 antibody, the LG displayed the capability of photothermal sensitivity and immunoregulation [121].Upon intertumoral injection, the LG precursor underwent a reversible change of gel-to-sol transition when the ambient temperature rose to 39 °C.The drug reservoir located in close proximity to the tumor site demonstrated persistent drug release, regulated by temperature fluctuations.This approach was subsequently validated for its ability to enhance the infiltration of lymphocytes into solid tumors of diverse origins.This approach effectively integrates the benefits of photothermal therapy and immunotherapy, capitalizing on the inherent low toxicity and biocompatibility of fundamental materials.There is less worry regarding its safety, and it has been observed that the innate immune system has the ability to protect against foreign substances, such as lipid-derived nanoparticles.These nanoparticles have the potential to activate the complement system through both the conventional and alternative pathways [119].However, lipid-based biomaterials possess distinct characteristics that set them in cancer therapy.A recent study demonstrated the potential for drug delivery via the skin or the blood-brain barrier.The lipid-based nanotechnology, which is projected to account for more than 50% of all lipidbased nano formulations [121], will make more contributions in the near future.
In addition to the aforementioned common materials, it is worth noting that there are additional possibilities for the creation of nanoparticles.The exceptional biocompatibility and consistency batch-to-batch consistency of protein products suggest that they may be well-received for clinical translation [122].Cai's team took advantages of human serum albumin (HAS) to create nanoparticles, thereby capitalizing on its inherent benefits.HAS has been found to exhibit remarkable stability and solubility within living organisms.Previous studies have demonstrated its ability to selectively target and affect tumor cells, making it a promising candidate for drugs carrying [123].In this study, the HAS was served as a nanochaperone for the delivery of interleukin-12 (IL-12), the molecule that would otherwise induce severe systemic proinflammatory toxicity when systemic administrated.The IL-12 ultimately facilitated the secretion of CCL5, CLL2, and CXCL10, leading to substantial proliferation of CD8+ CAR-Ts.Beyond that, Wang et al. fabricated a unique nanoparticle derived from yeast cell wall, taking into account the potential contribution of microbes in activating the innate immune system and enhancing the effectiveness of the adoptive immunological response, so bolstering the antitumor immune reaction.Significant tumor regression was seen in melanoma-bearing mice with the utilization of PD-L1 inhibition.Nevertheless, the advancement of microbial-based cancer therapy is hindered by the presence of dose-dependent side effects, such as systemic toxicity [124].Irvine et al. developed protein nanogels, which were utilized for the delivery of IL-15 super-agonist.When conjugated with cell surface proteins, nanogels demonstrated the ability to unload drugs in a regulated manner upon receiving TCR signals during antigen recognition [125].

Inorganic materials
Inorganic materials have made tremendous achievements recent years and are promising to distinguish themselves in drug delivery or other biomedicine for their special biochemical signatures.
Porous inorganic biomaterials have the satisfactory large surface area, stability in in vivo environment and diverse architectures.As opposed to common inorganic material, silica, calcium carbonate demonstrates great biocompatibility and biodegradability by imitating internal biominerals [126,127].Further, CaCO 3 is capable of carrying multiple drugs simultaneously, which was called "molecular cocktail" by researchers [128].It is also found that Ca2+ could increase gene transfection efficacy when coprecipitated with DNA [129].
Graphene oxide is another attractive inorganic material.The 2-dimensional planar structure has won it considerable interest.Graphene-based structure is featured with unparalleled loading efficacy and acknowledged photosensitivity and conductivity, which guaranteed its position in material development [130,131].
Metal-organic frameworks (MOFs) are also burgeoning in drug delivery field.The scaffold possesses high storage capacity and readily manipulated for different functions.They could interplay with biological substance including bioactive cells.Zirconium MOFs have stood out amidst MOFs these days and are valued for both chemical and mechanical stability thanks to stronger Zr-carboxylate bonds and excellent porosity compared with remaining MOFs [132].It is believed that MOFs will play a role in gene delivery in the near future.

Scaffold-based systems
Since the first introduction in 1954, there has been a significant surge in research focused on the development of hydrogels.The hydrogel is well recognized as an exceptional biomaterial due to its numerous advantages.Firstly, hydrogel is a 3D structure comprised of hydrophilic polymers, which can be either of natural or synthetic origin.It has a high capacity for water absorption with ideal biodegradability, biocompatibility, and neglectable cellular toxicity.Secondly, it provides accurate and controlled release of substances, such as chemotherapy drugs or adjuvant agents (e.g., cytokines, antigens), in response to specific environmental trigger factors.These triggers can include external stimuli like mechanical forces, temperature, and magnetic fields, as well as internal stimuli such as pH, reactive oxygen concentration, and enzymatic activity.This phenomenon is sometimes referred to as its "reversible behavior".In the context of hydrogels, it has been shown that the retention duration is extended, leading to an expansion of multifunctional therapeutic performance.This enhancement can be attributed to the distinctive material characteristics of hydrogels, which also contribute to improved mechanical qualities.Furthermore, hydrogel has a great degree of tenability in terms of sizes and delivery methods, which encompass injection, transdermal delivery, in situ implantation, and other approaches, thus reducing adverse effects associated with systemic administration.Ultimately, hydrogel is one of few materials capable of the 3D models applied in bioengineering.As one of paramount qualities making it indispensable, hydrogel 3D model could simulate the realistic TME while exhibiting traits, such as the stiffness and ductility, and environmental heterogeneity that closely resemble those of the TME.This could potentially make a significant contribution to the verification of safety for biomaterials relevant to contemporary cancer therapy [133][134][135].
Natural polymer hydrogel is predominantly beneficial due to its potential to safeguard cell viability while minimally impeding cell bioactivity.Simultaneously, they demonstrate the production of endogenous extracellular signals and ligands without the requirement for complex artificial integration.One of the obstacles that can be identified is the lack of diversity across batches, leading to a sense of dissatisfaction.Additionally, another challenge is the rather unpredictable nature of biodegradation [134].
Natural gellan gum (GG) was recently adopted as a novel approach for efficiently stimulating T cell subpopulations through antigen presentation.Derived from bacterial exopolysaccharide, GG has consistency across batches and biocompatibility.Prior studies mirrored that it had superior capability of cell capture, adhesion, and proliferation without expensive polymer modifications to improve cell entrapment, as compared to traditional hydrogel [136].Recently, it has been reported the successful development of GG-based tailorable nanoparticles system for T cell activation.With specific ligands on its surface, these nanoparticles were found to generate large quantities of CD4 + regulatory T cells and inhibit T cells exhaustion in vitro.Due to its stability upon rehydration and enormous water uptake capacity, npGG particles have the potential to participate in downstream processes by absorbing biologically relevant molecules and facilitating their controlled in situ release.However, the logical relationship between these factors is unclear.This study presented a novel perspective on the utilization of hydrogels in the context of CAR-T therapy [26].
Another example of polysaccharide-based systems is the fucoidan-based complex coacervate-laden injectable hydrogel (FPC2-IG).Fucoidan belongs to glycosaminoglycan, a group of materials that have garnered significant attention in the field of protein delivery system.Glycosaminoglycan-based delivery systems exhibit notable efficacy in augmenting protein bioactivity.Fucoidan, owing to its remarkable protein binding capability, particularly in relation to interleukin-2 (IL-2), along with its low immunogenicity and natural abundance, demonstrates significant promise.Besides, the complex coacervate is a liquid-liquid phase separation phenomenon discovered in marine species, whose secretion of water-soluble polyelectrolytes formulate coacervates.It is the water-immiscibility, microencapsulation capability that contribute to high-effective delivery and sustained retention of uploaded protein in case of enzymatic degradation upon arrival of tumor sites.The researches selected poly-l-lysine, which has been approved by the U.S. Food and Drug Administration, as a positive electrolyte coupled with negatively charged FPC to form stable complex coacervate.The resulting layer was then embedded in a pH-regulated injectable hydrogel (FPC2-IG).To provide further clarification, the FPC2-IG-IL-2 treatment ultimately boosts the proliferation of cytotoxic lymphocytes and decreases the presence of myeloid populations by extending the duration of IL-2 release and modifying TME [137].
The application of hyaluronic acid (HA) has also been documented.Gu et al. developed the crosslink procedure of hydrogel through ultraviolet, including an acrylate group into the hydrogel, as shown in Fig. 4A.The study involved the development of a product that combined IL-15 encapsulation with platelets conjugated with PD-L1 inhibitors.This product aimed to enhance the long-term storage of medications and improve the dispersion of CAR-Ts when implanted in the remaining cavity following tumor excision, with the objective of preventing tumor relapse [138].
Alginate is readily available in nature and possesses a consistent capacity for reproduction.It is produced by bacterial biosynthesis tends to exhibit superior quality.Additionally, it possesses excellent biocompatibility and has demonstrated biodegradation without causing any adverse effects on genetic material or cell viability, owing to its inert characteristics.The primary polymer structure exhibits a significant abundance of hydroxyl and carboxyl functional groups, which have garnered considerable interest among researchers in the field of biosynthesis due to their facile modifiability and hydrophilic nature.Alginate hydrogel exhibits both good oxidation tolerance and immune-stimulation.Thus, these materials are widely used in drug-delivery system establishment [139,140].Chao et al. created a metformin-loaded alginate hydrogel which exerted positive effects on both TME regulation as well as CAR-T metabolism and activation [141].Composite platform based on alginate hydrogel was also investigated to explore better way of in situ or ex vivo activation of CAR-Ts.Wang's team designed an injectable microporous hydrogel.This innovates platform provided a storage solution for CAR-Ts, utilizing a nanoporous material matrix to reverse T cells activation signal.In the presence of T cells abundant in thiol groups, azido-functionalized alginatebased bulk-phase gel network can give a controlled release of microparticles that are bounded with anti-CD3 and anti-CD28 to effectively activate T cells in a responsive manner.Cost reduction related to cumbersome and error-prone manufacture procedure and allocation could be lower because such platform stored and sheltered the CAR-Ts prior to their delivery to the intended site, ensuring optimal therapy concentration within the tumor [142].
An injectable and photocurable hydrogel composed of gelatin methacryloyl was also documented in a scholarly study.The gelatin methacryloyl underwent scaffold formation with appropriate exposure to blue light.Within this scaffold, CAR-Ts had demonstrated a distinct distribution and maintained their biological activity within the TME.The procedure of curing solely necessitated photo-crosslinking for priming.It was relatively gentle and harmless processing means, requiring neither complex surgical procedures nor intricate environmental adjustments, and exhibiting a low tolerance to faults [31].
Regarding artificial synthesized hydrogel, have greater control over customizing its physical features and associated functionalities by using adjuvants, maintaining the batch-to-batch stability.Enzymolysis and recognition sites mimicking natural extracellular matrix could be secured through chemical synthesis [133,143].
Employed for persistent vaccine delivery, polymer-nanoparticle (PNP) hydrogels have been currently discovered to have potential application in CAR-T therapy.The injectable and selfassembled PNP were shown to induce a transitory inflammatory microenvironment, leading to improved proliferation and activation of immune cells.As shown in Fig. 4B, Zhu et al. described a synergetic strategy by combination of nanozymes and CAR-Ts in solid tumors to remodel TME.Researchers also found that both proximal and distal tumors exhibited satisfactory tumor regression after administration, suggesting its potent abscopal effect to eradicate inaccessible tumors.Subsequent observation revealed that PNP hydrogels could prevent the passive diffusion of cytokines while facilitating active motility of CAR-Ts.It could conclude that this technique exhibits great promises for future development [44].By virtue of its excellent biocompatibility and adaptability, the collaboration between hydrogel and other materials holds significant potential for yielding unexpected gains.Hu et al. conducted a pioneering attempt to transform the live DCs into artificial APCs (aAPCs) which holds dominant responsibility for antigenpresenting and CAR-T activation.In this work, researcher opted for the utilization of a photo initiator known as 2-hydroxyl-4'-(2-hydroxyethoxy)-2-methylpropiophenone photoinitiator (I2959) and poly (ethylene glycol) diacrylate (PEG-DA) as the hydrogel materials to enhance direct membranous permeation in case of mechanical membrane disruption in original investigations.The hydrogel monomers underwent crosslinking upon exposure to UV radiation, forming the gelated DCs.The feasibility of storage by freezing and lyophilization and its offthe-shelf convenience was proved through a series of studies.The hydrogels were capable of loading customized antigen peptide and coupled with microparticles releasing immuneboosting cytokines.Moreover, researchers applied such methodology on autologous primary human DCs, and those constructions retained inceptive bioactivity and stability.The research opened up novel possibility for combination between natural and man-made hybrid material system [144].
Watson-Crick base pairing rules as underlying principle, the strict interaction nature of 4 nucleotides makes the programmable nanoscale assembly of 2D or 3D DNA structures possible.Under the guidance of Rothemund's "scaffolded DNA origami" approach, it becomes possible to achieve the accurate fabrication of specified 3D structures using DNA and probably helps the large-scale production in the foreseeable future.Pure nucleic acid generated material lack of desirable electrical, mechanical, or catalytic properties.Therefore, chemical modification is commonly employed to introduce extra functionality [145][146][147].Recently, scientists introduced such technology into CAR-T therapy augmentation.In an attempt to address the unfulfilled requirements for biocompatible conjugation platform that could integrate multiple biomolecules in harmony on surface, oligonucleotides were exploited to provide a solution.The brief synthetic DNA scaffolds was then immobilized on polymer particles fabricated with poly (lactic-co-glycolic acid) (PLGA) to form the biocompatible immune cell-engaging particles.Through the hybridization of cDNA strands, protein of variety was further coloaded and attached to respective scaffold population.Subsequent experiments confirmed that immune cell-engaging particles, a highly modularized platform, have multiple functions that are contingent upon the assembly of variant proteins.One notable example is its ability to present antigens in a manner that enables precise control over the activation of CAR-Ts, like an AND-gate mechanism.It is believed that such modular and versatile platform have a significant impact for immune regulation in clinical practice [148].
Comprised of series of microscale needles, microneedle emerge as promising drug delivery platform.Microneedles have been available in several designs including solid, hollow, dissolving and drug reservoir, with each shape being tailored for varied delivery principals.An instance of a dissolving microneedle can be manufactured using biodegradable materials such as PVP.Drugs are capable to be continually released in the planting area following the gradual dissolution of needles to increase bioactive concentration.In addition to its local-controllable flexibility, microneedles are malleable for stimuli-responsive potential by means of utilizing specific materials.According to different biochemical and physical signal condition, microneedle exhibits the ability to integrate environmental analysis and supportive drugs delivery into 1 structure.Compared with other drugs delivery platforms, microneedles handle drugs fluctuating in size and species over a wider range of medicines varying in size and species.The possible cargo options encompass small molecules, macromolecules, nano particles or immune cells, which can be utilized for various purposes such as anti-inflammation, antibacteria, immune regulation, or tissue targeting.Additionally, the sharp and minuscule tip of needle allows it to bypass external impeditive barrier for drugs to penetrate, while minimizing the associated pain, hence enhancing patient compliance.Yet, it is of note that the balance between porosity and critical parameters such as mechanical force and cargo capacity must be carefully evaluated [103,149,150].Gu's team developed a polymer porous microneedle (PMN) patch in order to showcase a CAR-T delivery vehicle that is a multipoint, scattered, yet evenly distributed administration, as shown in Fig. 5A [151].
In present studies, PLGA was employed as a structural material to provide adequate stiffness for the insertion of the therapeutic agent into the tumor.The pores were created after neutralization reaction to accommodate the CAR-T therapy.Through experimental results, it was found that PMN-mediated transport caused homologous mapping while intra-tumor injection led to cell restriction.The PMN patch also maintained the cell viability by sheltering CAR-T within the pores.Additionally, it effectively penetrated the external physical barrier of solid tumors and enhanced the distribution of cells [151].
Nitinol has a rich historical background in its extensive utilization as an implant, particularly in cardiovascular [152] and other related domains, owing to its exceptional biocompatibility.In recent years, its application has been extended to immune cell therapy.Stephan's team fabricated micropatterned nickel titanium (also known as nitinol) using magnetron sputtering technique.The nitinol material exhibits a well-defined geometry with a high resolution at micrometre scale.This characteristic enhances the predictability of cargo delivery kinetics when compared to the random pore network found in polymer-based scaffolds.In addition, it demonstrates superior space utilization rate and elasticity, stability, and shape memory.All of these satisfied the requirements for preventing blockage of the implanted lumen, taking into account external compressive force or unknown luminal abnormalities.In this research, the fibrin layer of nitinol with T cell stimulant signal molecules was involved when T cell infiltrated into engineered micropatterns.The thin film functioned as both the catalyst for CAR-T multiplication and the carrier for targeted transportation.Additionally, it acted as a tumor stent, impeding tumor growth and prolonging overall survival.Nevertheless, a dual-sided dilemma arose regarding the nondegradability of nitinol [153].
Mechanical changes in tissues influence immune cells, tuning their effector functions, with the potential to promote both adaptive and maladaptive responses [154].Immune cells exhibited stronger signaling responses and cytokine secretion on stiff surfaces than they did on softer surfaces [155].The mechanical properties of some materials can be adjusted by varying the number of monomers or the degree of crosslinking, giving us the ability to manipulate the mechanical properties of the extracellular matrix [156].Thus, through adjusting stiffness, we are capable to modulate immune cell activation [157].
There continue to exist a wide range of efficacy-enhancing methods that have been derived from diverse ideas an illustrative instance would be the utilization of a vaccine-like enhancer for CAR-T therapy.The researchers developed a novel class of ligands, known as amphiphile CAR-T ligands (amph-ligands), which can effectively target lymph nodes through conjugation with albumin-binding phospholipid polymers.Upon the entrance of lymph nodes, the ligand is transferred to APCs, triggering the secretion of cytokines which subsequently prime the T cells that possess the predetermined and engineered CAR on their ligands [158].Moreover, as shown in Fig. 5B, Tang et al. created a CAR-T-based live microrobot (M-CAR T) by decorating CAR T with immunomagnetic beads using click conjugation [159].There remain significant opportunities for further advancement in the development of biomaterials for CAR-T therapy, as a substantial number of these materials have not yet undergone clinical trials or achieved compliance with the good manufacturing practice standard.To yet, the valuable biosynthetic properties exhibited by many materials in medical research have not been effectively translated into immune therapy, specifically CAR-T therapy.

Advancements
In recent years, propelled by remarkable strides in biomaterials and cell engineering technology, investigators have increasingly directed their attention toward harnessing biomaterials to augment the effectiveness of cellular immunotherapy.These endeavors encompass a range of strategies, which include mitigating immune evasion, achieving precise, targeted cell delivery, bolstering the survival of engineered T cells, and even orchestrating synergistic combination therapies.Agarwalla et al. described an implantable Multifunctional Alginate Scaffold for T Cell Engineering and Release (MASTER) that streamlines in vivo CAR-T manufacturing and control distal tumor growth in a mouse xenograft model of lymphoma, as shown in Fig. 6A [160].This nuanced approach not only holds the promise of optimizing therapeutic efficacy but also attenuating systemic exposure, thereby presenting a noteworthy advancement in the field of immunotherapeutic intervention, as shown in Fig. 6B [161].Despite the notable achievements of CAR-T therapy in the management of hematologic malignancies, its efficacy in treating solid tumors remains limited.The advancements of biomaterials have allowed CAR-T therapy to emerge in the treatment of solid tumors.
Grosskopf et al. developed a simple-to-implement injectable hydrogel for the controlled co-delivery of CAR-Ts and stimulatory cytokines that improve efficacy of solid tumors therapy (Fig. 6C) [32].Furthmore, Zhou et al. engineered an injectable CAR-T gelatin methacryloyl hydrogel drug delivery system, which was abbreviated as i-GMD.Impressively, this construct exhibited heightened antitumor efficacy, yielding a substantial increase in the survival rates of mice bearing melanoma.Cumulatively, these efforts underscore the potential of biomaterial scaffolds to function as conduits for delivering immunomodulators or immune cells (Fig. 6D) [31].
In addition, biomaterials, as a widely employed drug delivery approach, have shown their distinct advantages in incorporating CAR-T therapy.A pertinent instance in this realm is provided by the research conducted by Yu Chao et al.The researchers developed the CAR-T-Met@gel system, a hydrogel scaffold proficient in codelivering both metformin and CAR-Ts (Fig. 6E) [141].By incorporating CAR-Ts into a freeze-dried alginate hydrogel harbouring metformin, a platform was established wherein therapeutic agents and cells were smoothly merged.The effectiveness of this approach was additionally corroborated through rigorous in vivo experimentation on murine gastric cancer models.Noteworthy achievements included not only curtailing postoperative tumor recurrence but also impeding the growth of distant neoplasms.

Conclusions and Perspective
Cellular immunotherapy represents a highly promising strategy for treatment of tumor, wherein activated immune cells are employed to identify and eliminate specific tumor cells with better efficacy compared to traditional treatment modalities.Nevertheless, the clinical implementation of cellular immunotherapy is hindered by the notable problems of low response rates and adverse effects.Furthermore, its efficacy in treating solid tumors has been suboptimal.
Biomaterials confer significant advantageous applications in the field of cellular immunotherapy.Within this domain, biomaterials demonstrate the potential to enhance drug delivery precision, maintain cellular vitality, enable controlled release dynamics, possess immunomodulatory prowess, and boost treatment resilience.Collectively, these attributes synergistically amplify the efficacy and viability of cellular immunotherapy.Biomaterials serve as adept vehicles for ferrying active constituents, such as immune cells, cytokines, or pharmaceutical agents, efficiently transporting them to targeted treatment sites.This orchestrated delivery augments therapeutic potency, which is an important factor of paramount significance in addressing solid tumors.In addition, biomaterials furnish a conducive environment that fosters cellular adhesion, proliferation, and differentiation.This nurturing environment facilitates the sustained cellular vitality, hence optimizing therapeutic impact.By establishing a propitious microenvironment, biomaterials adeptly extend the durability and effectiveness of cellular immunotherapy, sustaining its potency within the body over an extended period of time.By leveraging techniques that encompass shape molding and bio-3D printing, diverse treatment paradigms can be tailored to individual patients, thereby ushering in personalized therapeutic strategies with enhanced efficacy.

Fig. 1 .
Fig. 1. (A) Schematics of the hybrid AAV-Sleeping Beauty (AAV-SB) construct, SB100X mRNA electroporation and CAR T/NK/macrophage/iPSC generation, which formed 2 core components of mRNA AAV-SB joint engineering of stable therapeutic immune cells (MAJESTIC) system.Copyright © 2023, Lupeng Ye et al., under exclusive license to Springer Nature Limited [55].(B) A schematic overview of intracellular delivery by membrane permeabilization with photothermal nanofibers.Copyright © 2021, Ranhua Xiong et al., under exclusive license to Springer Nature Limited [69].

Fig. 3 .
Fig. 3. (A) Schematic of labeling strategy to monitor T cell distribution following transplantation in tumor-bearing mice.Copyright © 2020 Elsevier Ltd [105].(B) Schematic of the dual-modal nanoprobe loaded engineered T cells, which enables fluorescence and CT imaging for real-time monitoring of the cells in vivo.Copyright © 2020 Elsevier Ltd [106].

Fig. 4 .
Fig. 4. (A) Schematic of the tumor resection model and implantation of the engineered HA hydrogel, which was developed by a biodegradable hydrogel reservoir that encapsulates CAR-Ts targeting the human chondroitin sulfate proteoglycan 4. Copyright © 2021, Quanyin Hu et al., under exclusive license to Springer Nature Limited [138].(B) A synergetic strategy by combination of nanozymes and CAR-Ts in solid tumors for enhanced infiltration and effector function of CAR-Ts.Copyright © 2021, Wiley [162].

Fig. 6 .
Fig. 6. (A) Rapid MASTER-mediated CAR-T generation and therapy compared to conventional CAR-T therapy.Copyright © 2022, Pritha Agarwalla et al., under exclusive license to Springer Nature America, Inc [160].(B) Biomaterial scaffold niche in the area of cancer immunotherapy.Copyright © 2020 American Chemical Society [161].(C) Schematic illustration demonstrating our proposed delivery method for CAR-Ts to solid tumors compared to tradition intravenous approaches.Copyright © 2022 Abigail K. Grosskopf et al. [32].(D) Schematic of the injectable CAR-T delivery system.© 2022 Elsevier Ltd [31].(E) Schematic illustration of tumor resection and implantation of CAR-T@Met/gel.© 2023 Elsevier Ltd [141].

Table 1 .
Biomaterials application in gene transduction

Table 2 .
Biomaterials application in CAR-T imaging

Table 3 .
Partial biomaterials for increasing efficacy